Elio Polli

Neuro-glial differentiation of human bone marrow stem cells in vitro

Bone marrow (BM) is a rich source of stem cells and may represent a valid alternative to neural or embryonic cells in replacing autologous damaged tissues for neurodegenerative diseases. The purpose of the present study is to identify human adult BM progenitor cells capable of neuro-glial differentiation and to develop effective protocols of trans-differentiation to surmount the hematopoietic commitment in vitro. Heterogeneous cell populations such as whole BM, low-density mononuclear and mesenchymal stem (MSCs), and several immunomagnetically separated cell populations were investigated. Among them, MSCs and CD90+ cells were demonstrated to express neuro-glial transcripts before any treatment. Several culture conditions with the addition of stem cell or astroblast conditioned media, different concentrations of serum, growth factors, and supplements, used alone or in combinations, were demonstrated to alter the cellular morphology in some cell subpopulations. In particular, MSCs and CD90+ cells acquired astrocytic and neuron-like morphologies in specific culture conditions. They expressed several neuro-glial specific markers by RT-PCR and glial fibrillary acid protein by immunocytochemistry after co-culture with astroblasts, both in the absence or presence of cell contact. In addition, floating neurosphere-like clones have been observed when CD90+ cells were grown in neural specific media. In conclusion, among the large variety of human adult BM cell populations analyzed, we demonstrated the in vitro neuro-glial potential of both the MSC and CD90+ subset of cells. Moreover, unidentified soluble factors provided by the conditioned media and cellular contacts in co-culture systems were effective in inducing the neuro-glial phenotype, further supporting the adult BM neural differentiative capability.

Repeated sequences in human DNA

Abstract Human DNA has been fractionated in Ag + Cs 2 SO 4 and Hg 2+ Cs 2 SO 4 preparative density gradients, and the fractions obtained have been centrifuged in neutral CsCl after extensive dialysis to eliminate Hg 2+ and Ag 2+ . By centrifugation in Ag + Cs 2 SO 4 a new satellite, called satellite DNA II, has been isolated from human DNA. It has a density of 1.693 g/ml. in neutral CsCl, accounts for 2% of the total approximately, renatures rapidly and separates into complementary strands having different densities in alkaline CsCl. In Hg 2+ Cs 2 SO 4 gradients human DNA appears to be composed of two classes of molecules. The first, which accounts for approximately 80% of the total, is highly heterogeneous in base composition, its density in CsCl ranging from 1.690 to 1.720 g/ml., and is distributed in Hg 2+ Cs 2 SO 4 so that the A·T-rich fractions are on the heavy side and the G·C-rich fractions on the light side, as expected on the basis of the preferential binding of Hg 2+ to A·T pairs. The second class, which accounts for approximately 15% of the total, is more homogeneous, has a density of 1.696 g/ml., and is located on the light side of the DNA band in the Hg 2+ Cs 2 SO 4 gradient. This suggests that the amount of Hg 2+ bound to this A·T-rich DNA is abnormally low. This second class of DNA has been isolated by preparative CsCl centrifugation from a pool of the light fractions obtained from DNA-Hg 2+ Cs 2 SO 4 centrifugation. It tends to renature after heat-denaturation, as shown by the shift of its density towards the native value in neutral CsCl.

Stem-cell therapy for amyotrophic lateral sclerosis

CONTEXT With the lack of effective drug treatments for amyotrophic lateral sclerosis (ALS), and compelling preclinical data, stem-cell research has highlighted this disease as a candidate for stem-cell treatment. Stem-cell transplantation is an attractive strategy for neurological diseases and early successes in animal models of neurodegnerative disease generated optimism about restoring function or delaying degeneration in human beings. The restricted potential of adult stem cells has been challenged over the past 5 years by reports on their ability to acquire new unexpected fates beyond their embryonic lineage (transdifferentiation). Therefore, autologous or allogeneic stem cells, undifferentiated or transdifferentiated and manipulated epigenetically or genetically, could be a candidate source for local or systemic cell-therapies in ALS. STARTING POINT Albert Clement and colleagues (Science 2003; 302: 113-17) showed that in SOD1G93A chimeric mice, motorneuron degeneration requires damage from mutant SOD1 acting in non-neuronal cells. Wild-type non-neuronal (glial) cells could delay degeneration and extend survival of mutant-expressing motorneurons. Letizia Mazzini and colleagues (Amyotroph Lateral Scler Other Motor Neuron Disord 2003; 4: 158-61) injected autologous bone-marrow-derived stem cells into the spinal cord of seven ALS patients. These investigators reported that the procedure had a reasonable margin of clinical safety. WHERE NEXT? The success of cell-replacement therapy in ALS will depend a lot on preclinical evidence, because of the complexity and precision of the pattern of connectivity that needs to be restored in degenerating motoneurons. Stem-cell therapy will need to be used with other drugs or treatments, such as antioxidants and/or infusion of trophic molecules.

Transplantation of undifferentiated human mesenchymal stem cells protects against 6-hydroxydopamine neurotoxicity in the rat.

Stem cells have been increasingly recognized as a potential tool to replace or support cells damaged by the neurodegenerative process that underlies Parkinsons disease (PD). In this frame, human adult mesenchymal stem cells (hMSCs) have been proposed as an attractive alternative to heterologous embryonic or neural precursor cells. To address this issue, in this study we implanted undifferentiated hMSCs into the striatum of rats bearing a lesion of the nigrostriatal pathway induced by local injection of 6-hydroxydopamine (6-OHDA), a widely recognized rodent model of PD. Before grafting, cultured hMSCs expressed markers of both undifferentiated and committed neural cells, including nestin, GAP-43, NSE, β-tubulin III, and MAP-2, as well as several cytokine mRNAs. No glial or specific neuronal markers were detected. Following transplantation, some hMSCs acquired a glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. More importantly, rats that received the striatal graft showed increased survival of both cell bodies and terminals of dopaminergic, nigrostriatal neurons, coupled with a reduction of the behavioral abnormalities (apomorphine-induced turning behavior) associated with the lesion. No differentiation of the MSCs toward a neuronal (dopaminergic) phenotype was observed in vivo. In conclusion, our results suggest that grafted hMSCs exert neuroprotective effects against nigrostriatal degeneration induced by 6-OHDA. The mechanisms underlying this effect remain to be clarified, although it is likely that the acquisition of a glial phenotype by grafted hMSCs may lead to the release of prosurvival cytokines within the lesioned striatum.

Mesenchymal stem cell transplantation for neurodegenerative diseases.

Neurodegenerative diseases are characterized by a progressive degeneration of selective neural populations. The lack of effective treatment and the characteristic of their pathology make these diseases appropriate candidates for cell therapy. Mesenchymal stem cells (MSCs) are multipotent stem-like cells that are capable of differentiating into mesenchymal and nonmesenchymal lineages. Their regenerative capacity after in vivo transplantation into animal models of neurodegenerative diseases has suggested that they could be useful against human diseases. Human bone marrow-derived MSCs (hMSCs) can be easily amplified in vitro and their transdifferentiation has been claimed in vitro and in vivo in neural cells. There are some doubts concerning the exact mechanisms responsible for the beneficial outcome observed after MSC transplantation into neurodegenerating tissues. Possible interpretations include cell replacement, trophic factor delivery, and immunomodulation. This review mainly concerns hMSCs transplantation in neurodegenerative diseases, because it has proven to be feasible, safe, and potentially effective. Although they have been used in hundreds of clinical trials, mixed results and no functional and long-lasting integration have so far been observed. hMSCs transplantations therefore still have their “dark side.” However, the challenge in well-planned clinical trials merits discussion.

Multiple neurogenic and neurorescue effects of human mesenchymal stem cell after transplantation in an experimental model of Parkinson's disease

Stimulation of endogenous repair in neurodegenerative diseases, such as Parkinsons disease (PD), appears to be a novel and promising therapeutic application of stem cells (SCs). In fact SCs could propel local microenvironmental signals to sustain active endeavors for damaged neurons substitution, normally failing in non-supportive pathological surroundings. In this study, we demonstrated that two different doses of naïve human adult mesenchymal stem cells (hMSCs), implanted in the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA), positively survived 23 days after transplantation. Their fate was directly influenced by the surrounding host environment while grafted hMSCs, dose dependently, regionally sustained the survival of striatal/nigral dopaminergic terminals and enhanced neurogenesis in the Subventricular Zone (SVZ). The number of proliferative cells (Ki67/Proliferating Cell Nuclear Antigen +) as well as neuroblasts migration significantly augmented in the lesioned striatum of transplanted animals compared to controls. No SVZ astrogenesis was detected in all experimental conditions, irrespectively of graft presence. Activation of endogenous stem cell compartments and rescue of dopaminergic neurons, supported by the persistent release of specific cytokine by MSCs in vivo, appeared in principle able to contrast the neurodegenerative processes induced by the 6-OHDA lesion. Our results suggest that reciprocal influences between grafted cells and endogenous neural precursors could be important for the observed neurorescue effect on several brain regions. Altogether, our data provide remarkable cues regarding the potential of hMSCs in promoting endogenous reparative mechanisms that may prove applicable and beneficial for PD treatment.

Hypertension in Man with a Kidney Transplant: Role of Familial versus Other Factors

Genetic factors are clearly involved in the pathogenesis of essential hypertension in man. In at least three rat models of genetic hypertension it is possible to transplant the hypertension with the kidney. To see whether or not the same is true for humans, we carried out a 2-year retrospective study of 50 selected recipients of a cadaver kidney. We correlated the following factors by multivariate statistical analyses: presence or absence of hypertension in the family of donor and recipients; donors and recipients age; mean blood pressure (MBP) and antihypertensive therapy (AHT) score during dialysis; months of dialysis and body surface before transplantation; body weight, plasma creatinine, prednisone dosage and cumulative rejections with MBP and AHT score at various time intervals after transplantation. The results obtained showed that, considering the recipients coming from normotensive families, the AHT score after transplantation was significantly greater (p less than 0.05 1st and p less than 0.01 2nd year) in the patients receiving a kidney removed from donors with hypertensive families than in patients receiving a kidney removed from donors with normotensive families. This difference was not present when the recipients coming from hypertensive parents were considered. AHT score after transplantation is also correlated with AHT score on dialysis (p less than 0.01 1st and 2nd year), body weight (p less than 0.02 1st and p less than 0.01 2nd year), cumulative rejections (p less than 0.025 1st and 2nd year) and inverse MBP after dialysis (p less than 0.025 2nd year).

Induction of neurotrophin expression via human adult mesenchymal stem cells: implication for cell therapy in neurodegenerative diseases.

In animal models of neurological disorders for cerebral ischemia, Parkinsons disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFRp75) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophins expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders.

Renaturation properties and localization in heterochromatin of human satellite DNA's

Abstract Human DNA has been fractionated by centrifugation in an Ag + -Cs 2 SO 4 preparative density gradient. Besides satellite DNA I and II, previously demonstrated and characterized, a newly identified satellite DNA III has been isolated, having a CsCl density of 1.696 g/ml and accounting for 1.5 % of the total genome. The renaturation properties of human satellite DNA III, estimated by determining its CsCl densities and melting curves after denaturation and renaturation, indicate that it is fast renaturing and therefore highly repeated, as are the other human satellite DNAs. The nuclei obtained from human placenta and leukemic leucocytes have been fractionated into heterochromatin and euchromatin. Satellite DNAs are enriched in heterochromatin, while they are no longer detectable in the DNA extracted from euchromatin, centrifuged in Ag + -Cs 2 SO 4 .

Globin gene deletion in HPFH, δ°β° thalassaemia and Hb Lepore disease

THE thalassaemias are a group of inherited disorders characterised by the defective production of either α (α thalassaemias) or non-α (β and δ°β° thalassaemias) globin chains of haemoglobins (Hb)1. In β thalassaemias the decreased synthesis of β-globin chains is only partially compensated by the increased production of γ chains, which probably reflects2 the massive hypertrophy of the erythron with selective survival of the clones of adult haemoglobin F-producing cells (F cells3,4). The situation is very different in other genetic disorders of the non-α gene cluster, known as δ°β° thalassaemias and Negro type of hereditary persistence of fetal haemoglobin (HPFH). In these two forms there is a genuine increase of γ-chain production, as shown by the high level of HbF found in heterozygotes. Although a clearcut distinction from both the clinical and haematological point of view cannot be traced between these two forms, the HPFH differs from the δ°β° thalassaemia in having a higher degree of γ-chain synthesis and a more homogeneous distribution of HbF within red cells. Recently, it has been possible to carry out gene analysis on DNA prepared from β°, δ°β° thalassaemic and HPFH patients. The β-globin gene is present in β° thalassaemias5–9, but in δ°β° thalassaemias and HPFH a major deletion, possibly involving both δ and β genes, has been demonstrated by hybridisation studies8,10–12. To characterise the molecular defect in these genetic disorders more precisely, we have hybridised DNA from homozygotes with HPFH, δ°β° thalassaemia and Hb Lepore disease (in which non-α-chains are a δβ fusion product13). For this we used a pure full-size cDNAβ probe and specific 5′ end and 3′ end cDNA fragments (we designate as 5′ end cDNA the portion corresponding to the 5′ end of the mRNA; the same for the 3′ end). Our results, reported here, show that in contrast to HPFH, where a complete δ and β gene deletion occurs, in δ°β° thalassaemia a 5′-end fragment of the δ gene is present.